7.1- using gene sequencing

Question 1

what is a genome?

Answer 1

total of all genetic material in an organism

Question 2

what does PCR stand for?

Answer 2

-polymerase chain reaction

Question 3

what is PCR

Answer 3

• a technique for amplification of DNA in vitro (outside of body)

Question 4

how does PCR work? 4 steps

Answer 4

1.DNA polymerase (duplicates DNA, taq is from Thermus aquaticus which is found in hot springs and is thus stable at high temperatures)
2. 2 primers (piece of single stranded DNA which is complementary to the specific target sequence at the 3’ end of each DNA replicated strand) This allows you to amplify any – enabling to find a needle in a haystack of DNA
3. free nucleotides to make amplified DNA
4.original DNA strand needed to be replicated

Question 5

PCR process summary

Answer 5

cycle
1.DNA heated to 95, h bonds between chains break, separate into 2 strands
2.mixture cooled to 50-65, allows primers to anneal/ attach to 3’ end of each strand
3.heated to 72 for DNA polymerase to attach nucleotides, Heat tolerant DNA polymerase replicates the region of DNA + takes longer for polymerisation of nucleotides
4. repeated cycle of heating and cooling, amplify region of DNA by thermal cycler 30times

Question 6

3 words which describe steps of PCR

Answer 6

1. denaturation- heat applied to template strand to separate double stranded DNA
2. annealing- temp decreased to primers can attach to 3’ end of each strand
   3.extension- primers extend from 5’ to 3’

Question 7

exons Vs introns

Answer 7

-exons are coding regions of DNA (genes)
-introns are large non coding regions of DNA that are removed before mRNA is translated into proteins

Question 8

how do we sequence DNA?

Answer 8

1. DNA chopped in to smaller pieces
   2.double strands separated into single
   3.PCR used to amplify DNA
   4.terminator bases are added to the strands
2. tags allow bases to be read rapidly

Question 9

what are terminator bases?

Answer 9

-modified versions of the bases (A,T,G,C)
-they have a fluorescent tag attached
-when incorporated into DNA, a terminator base stops anymore bases from being added

Question 10

what happens when the segments of DNA have been sequenced?

Answer 10

-raw data is fed into a computer system that reassembles the genome, by looking at overlap between fragments

Question 11

how can DNA sequencing be used to predict amino acid sequence?

Answer 11

-analysing base pairs enables us to work out which amino acids will be joined together to forma protein as a result of info contained in DNA
-predict which genes code for which proteins and how amino acids may join together in a polypeptide chain

Question 12

DNA sequencing links to disease management

Answer 12

-many diseases are the result of inheritance of a number of alleles or gene variants that increase risk of an individual developing a specific disease such as coronary heart disease or diabetes
-look at mutations which cause disease, then work out how the mutation in the protein then causes symptoms

Question 13

what does DNA profiling mean?

Answer 13

• we can identify individuals and species by patterns in their DNA
  -useful for forensic or paternity testing
  -process of identifying DNA variations for the purpose of identification

Question 14

what are genetic markers?

Answer 14

-regions of DNA which usually vary between individuals, used to construct genetic profiles

Question 15

where are differences in DNA generally found?

Answer 15

• introns (non coding regions)

Question 16

what are STRs?

Answer 16

-short sequences of DNA which are repeated many times within the introns, we call them micro or mini satellites

Question 17

micro Vs mini satellites

Answer 17

mini- 10-100 base sequences, repeated 50- several hundred times
micro- 2-6 bases repeated 5-100 times

Question 18

satellites

Answer 18

-found in same positions on each pair of chromosomes
-number of repeats between individuals changes, depending on what you inherit from your mother and father

Question 19

stage 1- how is a DNA profile produced?

Answer 19

-strands of DNA are cut into fragments using restriction endonucleases
-cut DNA at particular points in intron sequences known as recognition sites
-this gives repeated sequences that are intact, leaving a mixture of different sized DNA fragments

Question 20

stage2- gel electrophoresis

Answer 20

-separates DNA fragments so they can be identified
-DNA fragments placed in wells in agarose gel medium in buffering solution (to maintain constant pH) with known DNA fragments to aid identification
-gel contains dye that binds to DNA fragments in the gel and fluoresces under UV
-electric current passed through, DNA moves towards pos anode as slightly neg charge on phosphate group
-fragments move at diff rates according to size and charge

Question 21

stage 3- southern blotting

Answer 21

-alkaline buffer solution added and nylon filter paper placed over it, drawing DNA fragments to filter paper as blots
-the alkaline solution also denatures DNA fragments so strands separate and base sequences exposed